Scott L. Diamond

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9–ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9–ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Selectin-Like Kinetics and Biomechanics Promote Rapid Platelet Adhesion in Flow: The GPIbα-vWF Tether Bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb(alpha)) and the A1 domain of von Willebrand factor (vWF-A1). To date, limited information exists on the kinetics that govern platelet interactions with vWF in hemodynamic flow. We now report that the GPIb(alpha)-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of hydrodynamic flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k(0)(off) (3.45 +/- 0.37 s(-1)). Values for k(off), as determined by pause time analysis of transient capture/release events, were also found to vary exponentially (4.2 +/- 0.8 s(-1) to 7.3 +/- 0.4 s(-1)) as a function of the force applied to the bond (from 36 to 217 pN). The biological importance of rapid bond dissociation in platelet adhesion is demonstrated by kinetic characterization of the A1 domain mutation, I546V that is associated with type 2B von Willebrand disease (vWD), a bleeding disorder that is due to the spontaneous binding of plasma vWF to circulating platelets. This mutation resulted in a loss of the shear threshold phenomenon, a approximately sixfold reduction in k(off), but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb(alpha)-vWF-A1 tether bond that in part may explain the preferential binding of platelets to vWF at sites of vascular injury, the lack of spontaneous platelet aggregation in circulating blood, and a mechanism to limit thrombus formation.

Comparison of PSGL-1 Microbead and Neutrophil Rolling: Microvillus Elongation Stabilizes P-Selectin Bond Clusters

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophils surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.

Inner clot diffusion and permeation during fibrinolysis.

A model of fibrinolysis was developed using multicomponent convection-diffusion equations with homogeneous reaction and heterogeneous adsorption and reaction. Fibrin is the dissolving stationary phase and plasminogen, tissue plasminogen activator (tPA), urokinase (uPA), and plasmin are the soluble mobile species. The model is based on an accurate molecular description of the fibrin fiber and protofibril structure and contains no adjustable parameters and one phenomenological parameter estimated from experiment. The model can predict lysis fronts moving across fibrin clots (fine or coarse fibers) of various densities under different administration regimes using uPA and tPA. We predict that pressure-driven permeation is the major mode of transport that allows for kinetically significant thrombolysis during clinical situations. Without permeation, clot lysis would be severely diffusion limited and would require hundreds of minutes. Adsorption of tPA to fibrin under conditions of permeation was a nonequilibrium process that tended to front load clots with tPA. Protein engineering efforts to design optimal thrombolytics will likely be affected by the permeation processes that occur during thrombolysis.

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X = all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXaβ, factor XIa and factor α XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates × 24 different proteases × 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1 = Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.

Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear‐resistance of platelet aggregates

Summary.  Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1–10 mL of blood and are poorly suited for murine whole blood experiments. Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 μL of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid‐soluble collagen in 100 μm × 100 μm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti‐mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin α2 subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a ‘shear step‐up’ to 8000 s−1. Results: Wild‐type murine platelets adhered and aggregated on collagen in a biphasic shear‐dependent manner with increased deposition from 100 to 400 s−1, but decreased deposition at 1000 s−1. Adhesion to patterned collagen was severely diminished for platelets lacking a functional α2β1 integrin. Those integrin α2‐deficient platelets that did adhere were removed from the surface when challenged to shear step‐up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step‐up. Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin‐dependent pathways such as fibrin formation.

Printing chemical libraries on microarrays for fluid phase nanoliter reactions

Chemical compounds within individual nanoliter droplets of glycerol were microarrayed onto glass slides at 400 spots/cm2. Using aerosol deposition, subsequent reagents and water were metered into each reaction center to rapidly assemble diverse multicomponent reactions without crosscontamination or the need for surface linkage. This proteomics technique allowed the kinetic profiling of protease mixtures, protease–substrate interactions, and high-throughput screening reactions. An inhibitor of caspases 2, 4, and 6 was identified by using a 352-compound combinatorial library microarrayed in quadruplicates on 100 slides and screened against caspases 2, 4, and 6, as well as thrombin and chymotrypsin. From one printing run that consumes <1 nanomole of each compound, large combinatorial libraries can be subjected to numerous separation-free homogeneous assays at volumes 103–104 smaller than current high-throughput methods.

SHEAR STRESS INDUCTION OF THE ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE IS CALCIUM-DEPENDENT BUT NOT CALCIUM-ACTIVATED

Arterial levels of shear stress (25 dynes/cm2) can elevate constitutive endothelial nitric oxide synthase (eNOS) gene expression in cultured endothelial cells(Ranjan et al., 1995). By PhosphorImaging of Northern blots, we report that the eNOS/glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) ratio in bovine aortic endothelial cells (BAEC) increased by 4.8‐ and 7.95‐fold after 6‐hr shear stress exposure of 4 and 25 dynes/cm2, respectively. Incubation of BAEC with dexamethasone (1 μM) had no effect on shear stress induction of eNOS mRNA. Buffering of intracellular calcium in BAEC with bis‐(o‐aminophenoxy)‐ethane‐N,N,N′, N′‐tetraacetic acid, tetra(acetoxymethyl)‐ester (BAPTA/AM) reduced shear stress induction of eNOS mRNA by 70%. Yet, stimulation of BAEC with ionomycin (0.1–1.0 μM) for 6–24 hr to elevate intracellular calcium had no effect on eNOS mRNA. These studies indicated that the shear stress induction of eNOS mRNA was a calcium‐dependent, but not calcium‐activated, process. Shear stress was a very potent and rapid inducer of the eNOS mRNA, which could not be mimicked with phorbol myristrate acetate or endotoxin. Inhibition of tyrosine kinases with genistein (10 μM) or tyrphostin B46 (10 μM) or inhibition of G‐protein signaling with guanosine 5′‐O‐(2‐thiodiphosphate) (GDP‐βS) (600 μM, 6‐hr preincubation) did not block the shear stress elevation of eNOS mRNA. J. Cell. Physiol. 171:205–211, 1997.

Determination of surface tissue factor thresholds that trigger coagulation at venous and arterial shear rates: amplification of 100 fM circulating tissue factor requires flow

Protein microarrays presenting spots of collagen and lipidated tissue factor (TF) allowed a determination of the critical surface concentration of TF required to trigger coagulation under flow. Whole blood supplemented with corn trypsin inhibitor (to inhibit factor XIIa) was perfused over microarrays for 5 minutes. Immunofluorescence staining of platelet glycoprotein GPIbalpha and fibrin(ogen) revealed a critical TF concentration (EC50) of 3.6, 8.4, and 10.2 molecules-TF/microm2 at wall shear rates of 100, 500, and 1000 s(-1), respectively. For collagen arrays where only the center lane of spots (in the direction of flow) contained TF, a downstream distance of 14 mm was required for the thrombus to widen enough to reach across a 300-micrometer gap to the adjacent TF-free lanes of collagen spots, in agreement with numerical simulation. To investigate the effect of low levels of circulating TF, whole blood (+/-100 fM added TF) was tested under static and flow conditions. After 5 minutes, the addition of 100 fM TF to whole blood had negligible effect under static conditions, but caused a 2.5-fold increase in fibrin formation under flow. This report defines the threshold concentrations of surface TF required to trigger coagulation under flow.